Transgenic animals capable of being induced to delete senescent cells

ABSTRACT

This document relates to methods and materials involved in the removal of senescent cells within a mammal. For example, transgenic non-human animals that can be induced to delete senescent cells are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/125,841, filed Mar. 4, 2014, which is a National Stage applicationunder 35 U.S.C. §371 of International Application No. PCT/US2012043613,having an International Filing Date of Jun. 21, 2012, which claims thebenefit of U.S. Provisional Application Ser. No. 61/567,587, filed Dec.6, 2011 and U.S. Provisional Application Ser. No. 61/499,616, filed Jun.21, 2011. The disclosures of these prior applications are consideredpart of (and are incorporated by reference in) the disclosure of thisapplication.

BACKGROUND

1. Technical Field

This document relates to methods and materials involved in the removalof senescent cells within a mammal. For example, this document providestransgenic non-human animals that can be induced to delete senescentcells.

2. Background Information

Cellular senescence, which halts the proliferation of damaged ordysfunctional cells, is widely recognized as an important mechanism toconstrain the malignant progression of tumor cells (Campisi, Curr. Opin.Genet. Dev., 21:107-112 (2011); and Kuilman et al., Genes Develop.,24:2463-2479 (2010)). As cells senesce, they can develop a uniquephenotype, referred to as the senescence-associated secretory phenotype(SASP, or alternatively called SMS), in which they acquire the abilityto secrete a variety of growth factors, cytokines, chemokines, andproteases (Coppe et al., PLoS Biol., 6:2853-2868 (2008)). Theobservation that senescent cells can accumulate in several tissues andorgans during organismal aging and are present at sites of age-relatedpathologies has led to speculation that they contribute to aging andage-related dysfunction (Campisi, Cell, 120:513-522 (2005)).

SUMMARY

This document relates to methods and materials involved in the removalof senescent cells within a mammal. For example, this document providestransgenic non-human animals that can be induced to delete senescentcells (e.g., p16^(Ink4a)-positive senescent cells). As described herein,transgenic mice can be produced to contain nucleic acid that allows forthe controlled clearance of senescent cells (e.g., p16^(Ink4a)-positivesenescent cells) by controllably inducing apoptosis of senescent cellswhile inducing little, or no, apoptosis of non-senescent cells. Forexample, a transgenic non-human animal provided herein can be allowed togrow and develop for a period of time and then can be treated with acompound (e.g., AP20187) capable of inducing apoptosis of senescentcells within the transgenic animal while inducing little, or no,apoptosis of non-senescent cells within the transgenic animal. Asdescribed herein, clearance of senescent cells within a transgenicnon-human animal can delay or reduce the likelihood of age-relateddisorders and can maximize healthy lifespan. In some cases, a transgenicnon-human animal provided herein can include nucleic acid encoding amarker polypeptide (e.g., a fluorescent polypeptide such as a greenfluorescent protein (GFP)) configured to be expressed by senescent cellswith little, or no, expression by non-senescent cells. In some cases, atransgenic non-human animal provided herein can have a geneticbackground (e.g., a BubR1 hypomorphic (BubR1^(H/H)) genetic background)that results in a markedly shortened lifespan with or without exhibitingone or more age-related phenotypes such as infertility, lordokyphosis,sarcopenia, cataracts, fat loss, cardiac arrhythmias, arterial wallstiffening, impaired wound healing, and dermal thinning.

The transgenic non-human animals provided herein can be used in assaysdesigned to identify agents having the ability to kill, or to facilitatethe killing of, senescent cells. For example, transgenic non-humananimals provided herein can be used as controls (e.g., positivecontrols) for the successful clearance of senescent cells. In somecases, transgenic non-human animals provided herein can be used ascontrols (e.g., positive controls) for the successful clearance ofsenescent cells with minimal or no killing of non-senescent cells.

In some cases, transgenic non-human animals provided herein can be usedas test animals in assays designed to identify agents having the abilityto kill, or to facilitate the killing of, senescent cells. In suchcases, the ability of a test agent to kill, or to facilitate the killingof, senescent cells can be monitored based, at least in part, on theexpression of a marker polypeptide (e.g., a fluorescent polypeptide suchas GFP) configured to be expressed by senescent cells. In some cases,the ability of the test agent to kill, or to facilitate the killing of,senescent cells can be evaluated by comparing its effects in aparticular animal at a first time point to the effects observed in thesame animal after treatment with a compound (e.g., AP20187) capable ofinducing apoptosis of senescent cells within that transgenic animal at asecond time point. Such a comparison can be used to identify test agentsthat are less effective or at least as effective as the compound capableof inducing apoptosis of senescent cells at the second time point. Insome cases, the compound capable of inducing apoptosis of senescentcells can be used at the first time point, and the test agent can beused as the second time point to identify test agents that are moreeffective than the compound used at the first time point.

In some cases, the transgenic non-human animals provided herein can beused in assays designed to identify agents having the ability to delayor reduce the likelihood of age-related disorders and/or maximizehealthy lifespan. For example, transgenic non-human animals providedherein can be used as controls (e.g., positive controls) for thesuccessful delay of age-related disorders and/or for the successfulincreased duration of a healthy lifespan.

In some cases, transgenic non-human animals provided herein can be usedas test animals in assays designed to identify agents having the abilityto delay or reduce the likelihood of age-related disorders and/ormaximize healthy lifespan. In such cases, the ability of a test agent todelay or reduce the likelihood of age-related disorders and/or maximizehealthy lifespan can be monitored based, at least in part, on theexpression of a marker polypeptide (e.g., a fluorescent polypeptide suchas GFP) configured to be expressed by senescent cells. In some cases,the ability of the test agent to delay or reduce the likelihood ofage-related disorders and/or maximize healthy lifespan can be evaluatedby comparing its effects in a particular animal at a first time point tothe effects observed in the same animal after treatment with a compound(e.g., AP20187) capable of inducing apoptosis of senescent cells withinthat transgenic animal at a second time point. Such a comparison can beused to identify test agents that are less effective or at least aseffective as the compound capable of inducing apoptosis of senescentcells at the second time point. In some cases, the compound capable ofinducing apoptosis of senescent cells can be used at the first timepoint, and the test agent can be used at the second time point toidentify test agents that are more effective at delaying or reducing thelikelihood of age-related disorders and/or maximizing healthy lifespanthan the compound used at the first time point.

In general, one aspect of this document features a transgenic mouse, thenucleated cells of which contain a transgene. The transgene comprises,or consists essentially of, a promoter sequence operably linked to anucleic acid sequence encoding a polypeptide having the ability to killa cell or facilitate the killing of a cell when the transgenic mouse isadministered a compound, wherein senescent cells of the transgenic mouseexpress the polypeptide, and wherein the senescent cells of thetransgenic mouse are killed when the compound is administered to thetransgenic mouse. Less than 10 percent of non-senescent cells of thetransgenic mouse can be killed when the compound is administered to thetransgenic mouse. The promoter sequence can be a p16^(Ink4a) promotersequence. The polypeptide can comprise a caspase 8 polypeptide sequence.The polypeptide can comprise a FKBP polypeptide sequence. Thepolypeptide can be a FKBP-caspase 8 fusion polypeptide. The compound canbe AP20187. The genetic background of the transgenic mouse can be aBubR1^(H/H) genetic background. The transgene can comprise nucleic acidencoding a marker polypeptide. The marker polypeptide can be a GFPpolypeptide.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1. p16^(Ink4a)-specific expression of the INK-ATTAC transgene. A,Schematic representation of the INK-ATTAC transgenic construct and themechanism of apoptosis activation. B, GFP intensity of TAT collectedfrom 5-month-old untreated mice with the indicated genotypes. C, qRT-PCRanalysis of untreated 9-month-old mouse tissue analyzed for relativeexpression of p16^(Ink4a) (left), FKBP-Casp8 (middle), and EGFP (right).Error bars, s.d.; n=3 female mice per genotype. Abbreviation: SkM,skeletal muscle (gastrocnemius). D, Cultured bone marrow cells from2-month-old WT:INKATTAC-3 mice treated with rosiglitazone for 48 hoursand immunostained for Flag antibody (visualizing Flag-FKBP-Casp8). Scalebar, 20 μm.

FIG. 2. Validation of p16^(Ink4a)-specific expression of the INK-ATTAC-5transgene. A, GFP intensity of TAT collected from 5-month-old untreatedmice with the indicated genotypes. Scale bar, 20 μm. B, qRT-PCR analysisof untreated 9-month-old mouse tissue analyzed for the relativeexpression of p16^(Ink4a) (left), FKBP-Casp8 (middle), and EGFP (right).Error bars, s.d.; n=3 female mice per genotype. Abbreviation: SkM,skeletal muscle (gastrocnemius).

FIG. 3. Tissues expressing the INK-ATTAC transgene have high levels ofcellular senescence markers. A, SA-β-Gal staining of TAT in 9-month-olduntreated mice with the indicated genotypes. B, qRT-PCR analysis of theindicated tissues for selected markers of senescence. Tissues werecollected from 9-month-old untreated mice with the indicated genotypes.Abbreviation: SkM, skeletal muscle (gastrocnemius). C, RepresentativeFACS profile of single-cell suspensions of TAT reveals two distinct cellpopulations based on GFP intensity of untreated BubR1^(H/H):INK-ATTAC-3mice (dashed line indicates GFP⁻ versus GFP⁺ cells). D, Sorted GFP⁺cells from TAT express higher levels of senescence-associated genes thanGFP⁻ cells. For B and D: error bars, s.d.; n=3 female mice per genotype.

FIG. 4. Tissues expressing the INK-ATTAC-5 transgene display elevatedindicators of senescence. A, qRT-PCR analysis of the indicated tissuesfor selected markers of senescence. Tissues were collected from9-month-old untreated mice with the indicated genotypes. Abbreviation:SkM, skeletal muscle (gastrocnemius). B, Sorted GFP⁺ cells from IATexpress higher levels of senescence-associated genes than GFP⁻ cells.For A and B, error bars, s.d.; n=3 female mice per genotype.

FIG. 5. Delayed onset of p16^(Ink4a)-mediated age-related phenotypes inAP20187-treated BubR1^(H/H):INK-ATTAC mice. A, Bone marrow cellscollected from 2-month-old WT:INK-ATTAC-3 mice were exposed torosiglitazone for 5 days to induce senescence and then cultured in thepresence or absence of AP20187. After 48 hours, cells were stained forSA-β-Gal. Scale bar, 50 μm. B, Lordokyphosis (left) and cataracts(right) are significantly delayed when BubR1^(H/H):INK-ATTAC transgenicmice were continuously treated with AP20187 from weaning age on.Lordokyphosis: *, P<0.0001 and **, P=0.0077; cataracts: *, P=0.0017 and**, P=0.0158 log-rank tests. C, Representative images of 9-month-oldAP20187 treated (top) and untreated (bottom) BubR1^(H/H): INKATTAC-3mice. D, AP20187 treatment increases average muscle fiber diameter ofgastrocnemius (Gastro) and abdominal (ABD) muscles ofBubR1^(H/H):INK-ATTAC mice. Error bars, s.e.m.; n=6 mice per genotype.*, P=0.0157; **, P=0.0007; ***, P=0.0239, unpaired t tests. E, Durationof exercise to exhaustion reveals that AP20187-treatedBubR1^(H/H):INK-ATTAC mice have extended running time. Error bars, s.d.;n=6 mice per genotype; *, P=0.0236; **, P=0.0009, unpaired t tests. F,BubR1^(H/H):INK-ATTAC mice treated with AP20187 travel longer distanceson a treadmill. Error bars, s.d.; n=6 mice per genotype; *, P=0.0187;**, P=0.0012, unpaired t tests. G, AP20187-treated BubR1^(H/H):INK-ATTACmice exert more energy during exercise ability tests. Error bars, s.d.;n=6 mice per genotype; *, P=0.0065; **, P=0.0002, unpaired t tests. H,Sizes of various fat depots of BubR1^(H/H):INK-ATTAC transgenic mice areincreased in response to AP20187 treatment. Parentheses, s.d.; n=6 miceper genotype. Asterisks denote significant (P<0.05) changes compared tountreated animals of the same transgenic line, unpaired t tests.Abbreviations: POV, paraovarian; Peri, perirenal; Mes, mesenteric; SSAT,subscapular adipose tissue. Total fat percentage was determined by DEXAscanning I, Cell diameter of IAT in BubR1^(H/H):INK-ATTAC mice increasesin response to AP20187 treatment. Error bars, s.e.m.; n=6 female miceper genotype; *, P=0.0031; **, P=0.0003, unpaired t tests. J, Dermal andsubcutaneous adipose layer thickness of BubR1^(H/H):INK-ATTAC miceindicates consistently increased adiposity with treatment. Error bars,s.e.m.; n=6 female mice per genotype; *, P=0.0016 and **, P=0.0015,unpaired t tests.

FIG. 6. Age-associated traits of BubR1 hypomorphic mice that arep16^(Ink4a)-independent are not influenced by clearance ofp16^(Ink4a)-positive cells. A, The percentage of sinus pause rhythmdisturbances is similarly increased in both treated and non-treatedBubR1^(H/H):INK-ATTAC heart tissue. Abbreviation: BPM, beats per minute.B, Thinning of the aorta is not corrected by drug treatment inBubR1^(H/H):INK-ATTAC animals. Error bars, s.e.m.; n=6 female mice pergenotype. Consistent with this, p16^(Ink4a) and the INK-ATTAC transgeneare not expressed in this tissue. Error bars, s.d.; n=3. FIG. 6C is agraph plotting overall survival for the indicated mice.

FIG. 7. AP20187-treated BubR1^(14H):INK-ATTAC animals have reducednumbers of p16^(Ink4a)-positive senescent cells. A, SA-β-Gal staining ofIAT reveals that AP20187-treated adipose tissue attenuates the senescentphenotype driven by BubR1 hypomorphism. qRT-PCR analysis for indicatorsof senescence in IAT. B-D, Treatment of BubR1^(H/H):INK-ATTAC animalswith AP20187 leads to lower levels of senescence-associated markers inIAT (B), skeletal muscle (C), and eye (D). Error bars, s.d.; n=3 femalemice per genotype. E, BrdU incorporation rates as a measure ofreplicative senescence are elevated in IAT and skeletal muscle ofBubR1^(H/H):INK-ATTAC mice. Error bars, s.e.m.; n=6 mice per genotype.Statistical analysis was by unpaired t test: *, P=0.0146 and **,P=0.0137.

FIG. 8 contains graphs plotting the relative expression of the indicatedpolypeptides in IAT (A), skeletal muscle (B), and eye (C).

FIG. 9 is a listing of the nucleic acid sequence of a pBLUESCRIPT II KSvector containing a p16^(Ink4a)-ATTAC-IRES-GFP nucleic acid construct.

FIG. 10 is a listing of the nucleic acid sequence of FIG. 9 with thevarious vector components and construct components labeled.

DETAILED DESCRIPTION

This document relates to methods and materials involved in the removalof senescent cells within a mammal. For example, this document providestransgenic non-human animals that can be induced to delete senescentcells (e.g., p16^(Ink4a)-positive senescent cells). Such non-humananimals can be farm animals such as pigs, goats, sheep, cows, horses,and rabbits, rodents such as rats, guinea pigs, and mice, and non-humanprimates such as baboons, monkeys, and chimpanzees. The term “transgenicnon-human animal” as used herein includes, without limitation, foundertransgenic non-human animals as well as progeny of the founders, progenyof the progeny, and so forth, provided that the progeny retain thetransgene. The nucleated cells of the transgenic non-human animalsprovided herein can contain a transgene that includes a promotersequence (e.g., a p16^(Ink4a) promoter sequence) operably linked to anucleic acid sequence encoding a polypeptide capable of killing a cellor capable of facilitating the killing of a cell. A promoter sequence ofa transgene described herein can be one that drives polypeptideexpression in senescent cells while driving less, little, or noexpression in non-senescent cells. Examples of such promoters include,without limitation, a p16^(Ink4a) promoter sequence, a p21^(cip)promoter sequence, and a Pai1 promoter sequence.

In some cases, a polypeptide capable of killing a cell or capable offacilitating the killing of a cell can be a polypeptide that includestwo polypeptide sequences fused together (e.g., a fusion polypeptide).An example of such a fusion polypeptide can be a FKBP-caspase 8 fusionprotein. See, e.g., Pajvani et al., Nat. Med., 11:797-803 (2005). Otherexamples of polypeptides capable of killing a cell or capable offacilitating the killing of a cell that can be used as described hereininclude, without limitation, a FKBP-caspase-1 fusion polypeptide orFKBP-caspase-3 fusion polypeptide. In some cases, a polypeptide capableof killing a cell or capable of facilitating the killing of a cell canbe engineered to include a tag (e.g., a Flag tag). In some cases, atransgene provided herein can include nucleic acid encoding a markerpolypeptide such as a fluorescent polypeptide (e.g., GFP, BFP, or RFP).For example, a transgene provided herein can include nucleic acidencoding a polypeptide capable of killing a cell or capable offacilitating the killing of a cell followed by an internal ribosomeentry site followed by a marker polypeptide (e.g., GFP).

In some cases, a transgene can include a p16^(Ink4a) promoter sequencefollowed by nucleic acid encoding an FKBP-caspase 8 fusion protein. Insuch cases, administration of a compound such as AP20187 can result inapoptosis of cells expressing the FKBP-caspase 8 fusion protein. Forexample, senescent cells of a transgenic non-human animal providedherein can express the FKBP-caspase 8 fusion protein of a transgene byvirtue of the p16^(Ink4a) promoter sequence and can be selectively andcontrollably killed following administration of AP20187. AP20187 can beobtained as described elsewhere (U.S. Patent Application Publication No.2004/0006233).

The term “operably linked” as used herein refers to positioning aregulatory element (e.g., a promoter sequence, an inducible element, oran enhancer sequence) relative to a nucleic acid sequence encoding apolypeptide in such a way as to permit or facilitate expression of theencoded polypeptide. In the transgenes disclosed herein, for example, apromoter sequence (e.g., a p16^(Ink4a) promoter sequence) can bepositioned 5′ relative to a nucleic acid encoding a polypeptide (e.g.,an FKBP-caspase 8 fusion protein).

Various techniques known in the art can be used to introduce transgenesinto non-human animals to produce founder lines, in which the transgeneis integrated into the genome. Such techniques include, withoutlimitation, pronuclear microinjection (See, e.g., U.S. Pat. No.4,873,191), retrovirus mediated gene transfer into germ lines (Van derPutten et al., Proc. Natl. Acad. Sci. USA, 82:6148-1652 (1985)), genetargeting into embryonic stem cells (Thompson et al., Cell 56:313-321(1989)), electroporation of embryos (Lo, Mol. Cell. Biol., 3:1803-1814(1983)), and in vitro transformation of somatic cells, such as cumulusor mammary cells, followed by nuclear transplantation (Wilmut et al.,Nature, 385:810-813 (1997); and Wakayama et al., Nature, 394:369-374(1998)). For example, fetal fibroblasts can be genetically modified tocontain an INK-ATTAC construct (FIG. 1A), and then fused with enucleatedoocytes. After activation of the oocytes, the eggs are cultured to theblastocyst stage. See, for example, Cibelli et al., Science,280:1256-1258 (1998). Standard breeding techniques can be used to createanimals that are homozygous for the transgene from the initialheterozygous founder animals. Homozygosity is not required, however, asthe phenotype can be observed in hemizygotic animals.

Once transgenic non-human animals have been generated, expression of anencoded polypeptide (e.g., an FKBP-caspase 8 fusion protein or markerpolypeptide) can be assessed using standard techniques. Initialscreening can be accomplished by Southern blot analysis to determinewhether or not integration of the transgene has taken place. For adescription of Southern analysis, see sections 9.37-9.52 of Sambrook etal., 1989, Molecular Cloning, A Laboratory Manual, second edition, ColdSpring Harbor Press, Plainview; NY. Polymerase chain reaction (PCR)techniques also can be used in the initial screening. PCR refers to aprocedure or technique in which target nucleic acids are amplified.Generally, sequence information from the ends of the region of interestor beyond is employed to design oligonucleotide primers that areidentical or similar in sequence to opposite strands of the template tobe amplified. PCR can be used to amplify specific sequences from DNA aswell as RNA, including sequences from total genomic DNA or totalcellular RNA. Primers typically are 14 to 40 nucleotides in length, butcan range from 10 nucleotides to hundreds of nucleotides in length. PCRis described in, for example PCR Primer: A Laboratory Manual, ed.Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995.Nucleic acids also can be amplified by ligase chain reaction, stranddisplacement amplification, self-sustained sequence replication, ornucleic acid sequence-based amplified. See, for example, Lewis, GeneticEngineering News, 12:1 (1992); Guatelli et al., Proc. Natl. Acad. Sci.USA, 87:1874-1878 (1990); and Weiss, Science, 254:1292-1293 (1991).

Expression of a nucleic acid sequence encoding a polypeptide (e.g., anFKBP-caspase 8 fusion protein or marker polypeptide) in senescent cellsof transgenic non-human animals can be assessed using techniques thatinclude, without limitation, Northern blot analysis of tissue samplesobtained from the animal, in situ hybridization analysis, Westernanalysis, immunoassays such as enzyme-linked immunosorbent assays, andreverse-transcriptase PCR (RT-PCR). As described herein, expression ofan FKBP-caspase 8 fusion protein by senescent cells within thetransgenic animal can result in transgenic animals that can be treatedwith AP20187 such that the senescent cells are killed. Such transgenicanimals can exhibit delayed, or a reduced likelihood of, age-relateddisorders and/or a maximized healthy lifespan. It is understood that aparticular phenotype in a transgenic animal typically is assessed bycomparing the phenotype in the transgenic animal to the correspondingphenotype exhibited by a control non-human animal that lacks thetransgene.

A transgenic non-human animal provided herein can have any appropriategenetic background. In some cases, a transgenic non-human animalprovided herein can have a BubR1 hypomorphic (BubR1^(H/H)) geneticbackground, a Tert^(−/−) genetic background, or a bGF1⁺ (bovine growthhormone) genetic background.

This document also provides tissues (e.g., skin, eye, fat, muscle, lung,heart, bone, liver, intestine, kidney, spleen, brain, cartilage, marrow,adrenal glands, ovaries, and testes) and cells (e.g., fat cells,preadipocytes, skin or lung fibroblasts, muscle satellite cells,osteoblasts, bone marrow progenitor cells, neuronal progenitor cells,hepatocytes, endothelial cells, chondroblasts, and splenocytes cells)obtained from a transgenic non-human animal provided herein.

This document also provides methods for identifying agents having theability to kill, or to facilitate the killing of, senescent cells andmethods for identifying agents having the ability to delay, or reducethe likelihood of age-related disorders, and/or maximize healthylifespan. Such methods can include, for example, (1) targeting senescentcells based on compounds activated by enzyme activities that are higherin them than other cells (such as senescence-associated(3-galactosidase), such compounds killing the senescent cells uponactivation, (2) use of compounds that kill cells to which they bindthrough receptors that are more highly expressed by senescent than othercells (such receptors being identified by proteomic or expressionprofiling of senescent versus non-senescent cells or other approaches),or (3) compounds that are activated by metabolic processes that are moreactive in senescent than non-senescent cells (with such metabolicprocesses being identified through metabolomic, proteomic, expressionprofiling, or other means), with the compounds so activated killing thesenescent cell.

In some cases, methods for identifying agents having the ability tokill, or to facilitate the killing of, senescent cells and methods foridentifying agents having the ability to delay, or reduce the likelihoodof age-related disorders, and/or maximize healthy lifespan can includeobtaining senescent cells from a mammal (e.g., an animal model or ahuman). For example, a transgenic mouse provided herein such as atransgenic mouse that expresses a marker polypeptide (e.g., GFP) insenescent cells can be used to obtain senescent cells. Such a transgenicmouse can contain a transgene that includes a marker polypeptide (e.g.,GFP) operably linked to a promoter sequence that drives polypeptideexpression in senescent cells while driving less, little, or noexpression in non-senescent cells. Examples of such promoters include,without limitation, a p16^(Ink4a) promoter sequence, a p21cip promotersequence, and a Pai1 promoter sequence. The senescent cell can be anyappropriate cell type or from any appropriate tissue. For example,senescent cells can be obtained from fat or endothelial tissue. In somecases, senescent cells can be obtained from liver, bone marrow, heart,lung, or skin tissue.

Any appropriate method can be used to obtain senescent cells from amammal. For example, senescent cells expressing a marker polypeptide(e.g., GFP) under the control of a p16^(Ink4a) promoter sequence can beseparated from non-senescent cells using standard techniques such ascell sorting methods based on the expression of the marker polypeptide.In some cases, cell lines of senescent cells can be used in place offreshly obtained senescent cells to identify agents having the abilityto kill, or to facilitate the killing of, senescent cells and agentshaving the ability to delay, or reduce the likelihood of age-relateddisorders, and/or maximize healthy lifespan as described herein. In somecases, senescent cells can be obtained by cell passage in culture (e.g.,greater than about 12 to 15 cell passages for mouse embryonicfibroblasts and greater than about 20 cell passages at a 1:2 split ratiofor human cells) or by radiation treatment (e.g., treatment with about 5to about 50 Grays), a ceramide (e.g., C6, C16, or C18) treatment (e.g.,treatment with about 7 μM to about 15 μM (e.g., 13 μM) of ceramide suchas C16 for at least about 15 days), exposure to oncogenes or increasedexpression of oncogenes such as H-Ras or K-Ras (e.g., K-RasG12V),exposure to non-oncogenes or increased expression of non-oncogenes suchas JAK or STAT, or exposure to glucose (e.g., about 16.5 mM to about22.5 mM of D-glucose) for at least 10 days (e.g., greater than 30 days).In some cases, senescent cells can be obtained by exposing cells toreactive oxygen species or hydrogen peroxide to induce senescence via ap53 pathway.

Once obtained, the senescent cells can be exposed to a library of testagents individually or in pools to identify those agents or pools ofagents having the ability to kill, or to facilitate the killing of, thesenescent cells. Once identified as having the ability to kill, or tofacilitate the killing of, the senescent cells, the identified agent canbe applied to comparable non-senescent cells in comparableconcentrations to confirm that the agent has a reduced ability to kill,or to facilitate the killing of, non-senescent cells. Those agentshaving the ability to kill, or to facilitate the killing of, senescentcells with a reduced or no ability to kill, or to facilitate the killingof, non-senescent cells can be classified as being an agent having theability to delay, or reduce the likelihood of age-related disorders,and/or maximize healthy lifespan. In some cases, senescent cellsobtained from a transgenic mammal provided herein and treated in amanner that results in senescent cell death can be used as positivecontrols.

In some cases, an agent can be identified as having the ability to kill,or to facilitate the killing of, senescent cells or as having theability to delay, or reduce the likelihood of age-related disorders,and/or maximize healthy lifespan using in vivo techniques. For example,an animal model such as wild-type mice or animals, mice with a BubR1hypomorphic (BubR1^(H/H)) genetic background, or other mouse or animalmodels can be used. In such cases, a library of test agents can beadministered individually or in pools to the animals (e.g., mice), andthe animals (e.g., mice) can be assessed for indications that the testagent is capable of killing, or facilitating the killing of, senescentcells or is capable of delaying, or reducing the likelihood ofage-related disorders, and/or maximizing healthy lifespan. Indicationsof senescent cell killing or indications of delayed or reducedlikelihood of age-related disorders, and/or indications of maximizedhealthy lifespan can be detected and assessed as described herein. Forexample, the ability of an agent to increase the length of lifespan canbe assessed comparing treated and untreated mice with, for example, aBubR1 hypomorphic (BubR1^(H/H)) genetic background.

This document also provides methods and materials for identifyingmolecules (e.g., polypeptides, carbohydrates, lipids, and nucleic acids)possessed or expressed by senescent cells. For example, senescent cellscan be obtained as described herein and assessed to identify molecules(e.g., polypeptides) possessed or expressed by those senescent cells.Any appropriate method can be used to identify molecules possessed orexpressed by senescent cells. For example, polypeptide isolation andsequencing techniques can be used to identify polypeptides expressed bysenescent cells.

In some cases, a transgenic mouse provided herein can be used toidentify molecules (e.g., polypeptides and carbohydrates) possessed orexpressed by senescent cells. For example, a transgenic mouse providedherein can be treated with a compound (e.g., AP20187) starting at orbefore birth (e.g., shortly after fertilization via treatment of themouse's mother) such that senescent cells are killed or prevented fromdeveloping. In such cases, the resulting mouse can be immunologicallynaïve with respect to the molecules exclusively expressed by senescentcells. The immunologically naïve mouse can then be exposed to senescentcells or components from senescent cells (e.g., plasma membranes) in amanner designed to trigger an immune response. Resulting antibodies orantibody-producing cells can be isolated and assessed to confirm thatthe antibodies recognize a molecule presented or expressed by senescentcells. In some cases, the antibodies can be assessed for the ability tonot recognize molecules presented or expressed by non-senescent cells.Once such antibodies are obtained, they can be used to identify themolecule present or expressed by the senescent cells.

In some cases, antibodies directed to a molecule present or expressed bysenescent cells can be used to kill, or to facilitate the killing of,senescent cells or to delay, or reduce the likelihood of age-relateddisorders, and/or to maximize healthy lifespan. For example, antibodiesdirected to a molecule present or expressed by senescent cells can beconjugated with isotopes or toxins to form conjugates having the abilityto kill, or to facilitate the killing of, senescent cells or as havingthe ability to delay, or reduce the likelihood of age-related disorders,and/or maximize healthy lifespan.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

Examples Example 1 Clearance of p16^(Ink4a)-Positive Senescent CellsDelays Aging-Associated Disorders

To examine the role of senescence in aging and age-related pathologiesand to test whether elimination of senescent cells has beneficialeffects, a transgenic strategy that enabled clearance of senescent cellsin mice was designed. A 2617-bp fragment of the p16^(Ink4a) genepromoter, which is transcriptionally active in senescent, but notnon-senescent, cells (Wang et al., J. Biol. Chem., 276:48655-48661(2001)), was engineered into a nucleic acid construct upstream ofnucleic acid encoding a FKBP-caspase 8 fusion protein containing a Flagtag (Pajvani et al., Nat. Med., 11:797-803 (2005)) to create anINK-ATTAC construct (FIG. 1A). As shown in FIG. 1A, the INK-ATTACconstruct was designed to express the FKBP-caspase 8 fusion proteinwithin senescent cells. Once myristoylated, the FKBP-caspase 8 fusionprotein becomes membrane-bound, and addition of AP20187, a syntheticdrug, is capable of inducing dimerization of the membrane-boundmyristoylated FKBP-caspase 8 fusion protein, thereby inducing apoptosis.

In addition, an internal ribosome entry site (IRES) followed by an openreading frame coding for EGFP was added downstream of the nucleic acidencoding the FKBP-caspase 8 fusion protein (FIG. 1A). The nucleic acidencoding EGFP was added to allow for detection and collection ofp16^(Ink4a)-positive senescent cells. Injection of the resultingconstruct into fertilized eggs yielded nine transgenic INK-ATTAC founderlines.

To examine whether removal of p16^(Ink4a)-expressing cells istechnically feasible and whether this impacts age-associated deficits inmice, each of the founder lines were bred onto a BubR1 hypomorphic(BubR1^(H/H)) genetic background. BubR1^(H/H) mice have a markedlyshortened lifespan and exhibit a variety of age-related phenotypesincluding, without limitation, infertility, lordokyphosis, sarcopenia,cataracts, fat loss, cardiac arrhythmias, arterial wall stiffening,impaired wound healing, and dermal thinning (Baker et al., Nat. Genet.,36:744-749 (2004); Hartman et al., Neurobiol. Aging, 28:921-927 (2007);and Matsumoto et al., Stroke, 38:1050-1056 (2007)). BubR1^(H/H) mice canaccumulate p16^(Ink4a)-positive cells in several tissues in whichage-associated pathologies develop including, without limitation,adipose tissue, skeletal muscle, and eye.

To screen for transgene activity in p16^(Ink4a)-positive cells, samplesof inguinal adipose tissue (IAT) were collected from each of the nineBubR1^(H/H):INK-ATTAC strains at five months of age and analyzed for GFPexpression by fluorescence microscopy. GFP fluorescence was observed intwo of these strains, BubR1^(H/H):INK-ATTAC-3 andBubR1^(H/H):INK-ATTAC-5 (FIGS. 1B and 2A). Next, the extent to whichexpression of INK-ATTAC and endogenous p16^(Ink4a) overlap wasdetermined using a quantitative (q)RT-PCR approach. Consistent withearlier data from BubR1^(H/H) mice (Baker et al., Nat. Cell Biol.,10:825-836 (2008)), skeletal muscle, IAT, and eye exhibited increasedp16^(Ink4a) expression with aging (FIGS. 1C and 2B).BubR1^(H/H):INK-ATTAC-3 and BubR1^(H/H):INK-ATTAC-5 mice also exhibitedelevated INK-ATTAC and GFP transcript levels in these tissues. On theother hand, BubR1^(H/H) tissues in which p16^(Ink4a) is not induced,such as brain, lung, colon, liver, and heart, had no clear induction ofINK-ATTAC or GFP in BubR1^(H/H):INK-ATTAC-3 and BubR1^(H/H):INK-ATTAC-5mice (FIGS. 1C and 2B).

To confirm that transgenic INK-ATTAC and endogenous p16^(Ink4a) areunder the same transcriptional control mechanism outside the context ofBubR1 hypomorphism, bone marrow of wildtype (WT):INK-ATTAC transgeniclines 3 and 5 were harvested and cultured in the absence or presence ofrosiglitazone, a drug that can induce cellular senescence andp16^(Ink4a) expression through activation of PPARγ (Gan et al., J. CellSci. 121:2235-2245 (2008)) Immunofluorescence microscopy revealed that ahigh proportion of cells expressed Flag-tagged FKBP-Casp8 fusion proteinin the presence of rosiglitazone, but not in its absence (FIG. 1D).Together, these data indicated that INK-ATTAC gene activity in the twotransgenic founder lines overlaps with endogenous p16^(Ink4a)expression.

Next, the following was performed to determine whether INK-ATTAC isexpressed in senescent cells in BubR1 hypomorphic tissue. Fat tissue ofaged BubR1^(H/H):INK-ATTAC mice was strongly positive forsenescence-associated-β-galactosidase (SA-β-Gal; FIG. 3A). qRT-PCRanalysis demonstrated that INK-ATTAC expression correlates withexpression of senescence markers in IAT, including p21, Pai1, IL-6, andIgfbp2 (FIGS. 3B and 4A). Skeletal muscle and lens tissue of agedBubR1^(H/H):INK-ATTAC mice were SA-β-Gal negative, but both thesetissues expressed other markers of senescence, including Mmp13, Pai1,p21, and IL6 (FIGS. 3B and 4A). To obtain additional evidence forselective expression of INK-ATTAC in senescent cells, IAT was collectedfrom aged BubR1^(H/H):INK-ATTAC animals. Single-cell suspensions wereprepared by collagenase treatment, and GFP⁺ and GFP⁻ cell populationswere separated by fluorescence activated cell sorting (FACS; FIG. 3C).Each population was analyzed for expression of INK-ATTAC and senescencemarkers by qRT-PCR. GFP⁺ cells not only expressed much higher levels ofp16^(Ink4a) than GFP⁻ cells, but also exhibited elevated levels of otherkey senescence markers for IAT (FIGS. 3D and 4B).

Senescence markers in GFP⁻ cells from BubR1^(H/H):INK-ATTAC mice were aslow as in GFP⁻ cells from age-matched WT:INK-ATTAC mice. Taken together,these results indicated that INK-ATTAC is selectively expressed inp16^(Ink4a)-positive senescent cells.

To determine whether INK-ATTAC can eliminate senescent cells, bonemarrow cells of WT:INK-ATTAC transgenic lines 3 and 5 were cultured inthe presence of rosiglitazone to induce senescence, and cell survivalwas monitored after activating the FKBP-Casp8 fusion protein by AP20187treatment. The vast majority of cells from both transgenic lines werefound to be either dead or in the process of dying 48 hours after addingAP20187 (FIG. 5A). In contrast, parallel cultures that remaineduntreated consisted almost entirely of viable SA-β-Gal-positive cells.These data demonstrated that FKBP-Casp8 activation efficientlyeliminates p16^(Ink4a)-positive senescent cells in vitro.

The following was performed to examine whether clearance ofp16^(Ink4a)-expressing cells from BubR1^(H/H) mice prevents or delaysthe onset of age-related phenotypes in this progeroid background. Tothis end, cohorts of BubR1^(H/H):INK-ATTAC-3 and BubR1^(H/H):INK-ATTAC-5mice were established, which were either treated with AP20187 everythird day beginning at 3 weeks of age or left untreated. Both treatedand untreated mice were monitored for development of age-associateddeficits known to accompany p16^(Ink4a) induction, including sarcopenia,cataracts, and loss of adipose tissue (Baker et al., Nat. Cell Biol.,10:825-836 (2008)). Treated mice of both BubR1^(H/H):INK-ATTAC linesexhibited substantially delayed onset of lordokyphosis (a measure ofsarcopenia in this model) and cataracts compared to untreated mice,which developed these phenotypes at a rate similar to BubR1^(H/H) micelacking the INK-ATTAC transgene (FIGS. 5B and 5C). Consistent withdecreased lordokyphosis, muscle fiber diameters of AP20187-treatedBubR1^(H/H):INK-ATTAC animals were larger than those of untreatedcounterparts (FIG. 5D). In addition to muscle retention, treadmillexercise tests revealed that duration of exercise (FIG. 5E), distancetraveled (FIG. 5F), and overall amount of work performed (FIG. 5G) wereall significantly increased in the animals treated with AP20187,indicating preservation of muscle function. Dual-energy x-rayabsorptiometry (DEXA) scans of BubR1^(H/H):INK-ATTAC mice confirmed thatAP20187 treatment prevented loss of adipose tissue (FIG. 5H). All majorfat deposits were larger in AP20187-treated BubR1^(H/H):INK-ATTACanimals (FIG. 5H), and individual adipocytes were markedly increased insize (Figure SI). Consistent with this generally increased adiposity,dorsal skin contained significantly more adipose tissue (FIG. 5J).

Age-related phenotypes of BubR1^(H/H) mice that arise in ap16^(Ink4a)-independent fashion, such as cardiac arrhythmias andarterial wall stiffening (Matsumoto et al., Stroke, 38:1050-1056(2007)), were not attenuated in AP20187-treated BubR1^(H/H):INK-ATTAC-3and BubR1^(H/H):INK-ATTAC-5 mice (FIGS. 6A and 6B). This correlated withlack of INK-ATTAC induction in heart and aorta (FIGS. 1C and 6B).Cardiac failure is presumably the main cause of death in BubR1^(H/H)mice, which could explain why the overall survival of AP20187-treatedBubR1^(H/H):INK-ATTAC mice was not substantially extended (FIG. 6C). Toexamine whether clearance of p16^(Ink4a)-positive cells might have anyovertly negative side effects, WT:INK-ATTAC mice were continuouslytreated with AP20187 until eight months of age. No such overtly negativeside effects were observed. Taken together, these results indicated thatcontinuous removal of p16^(Ink4a)-expressing cells fromBubR1^(H/H):INK-ATTAC mice selectively delays age-related phenotypesthat depend on p16^(Ink4a) induction.

The following was performed to determine whether the delayed onset ofage-related pathologies coincided with a reduction in the number ofsenescent cells in these tissues. TAT of AP20187-treatedBubR1^(H/H):INK-ATTAC mice exhibited a dramatic decrease in SA-β-Galstaining compared with TAT of untreated counterparts (FIG. 7A).Corresponding decreases of other senescence-associated markers were alsoobserved, as well as expected reductions in INK-ATTAC and GFP (FIGS. 7Band 8A). Skeletal muscle (FIGS. 7C and 8B) and eye (FIGS. 7D and 8C)exhibited a similar reduction in senescence indicators. BrdUincorporation was lower in TAT and muscle tissue of untreated than intreated animals (FIG. 7E), further supporting the contention thatsenescence-associated replicative arrest is decreased uponadministration of AP20187 in BubR1^(H/H):INK-ATTAC transgenic animals.Together, these results indicated that senescent cells were cleared fromtissues and that this delays acquisition of age-related dysfunction inBubR1 hypomorphic mice.

The results provided herein demonstrate the generation of a transgenicmouse model that allows for the inducible removal ofp16^(Ink4a)-positive senescent cells. By breeding this model into aprogeroid mouse genetic background, the clearance ofp16^(Ink4a)-expressing senescent cells selectively was shown to delayonset of age-related pathologies in tissues that accumulate these cells,demonstrating that development of age-related pathologies and cellularsenescence are clearly linked in this model. These results alsodemonstrate that therapeutic interventions to clear senescent cells orblock their effects represent an avenue for treating or delayingage-related diseases and improving healthy human lifespan.

Methods and Materials

The INK-ATTAC transgenic construct was made as follows. The FKBP-Casp8fragment was subcloned from the aP2-ATTAC transgenic construct (Pajvaniet al., Nat. Med., 11:797-803 (2005)), and inserted into pBlueScriptII(Stratagene). A 2617-bp segment of the murine p16^(Ink4a) promoter wasPCR amplified from BAC DNA to replace the aP2 promoter. An IRES-EGFPfragment was inserted 3′ of the ATTAC. Nine transgenic founder lines ofmice were obtained by injection of this construct into FVB oocytes usingstandard methods. A PCR-based method was used for INK-ATTAC transgeneidentification. BubR1^(H/H) mice were generated as described elsewhere(Baker et al., Nat. Genet., 36:744-749 (2004)). For AP20187 (ARIADPharmaceuticals, Inc.; Cambridge, Mass.) treatments, animals wereinjected intraperitoneally (i.p.) every three days with 0.2 μg/g bodyweight of the dimer-inducing drug (Pajvani et al., Nat. Med., 11:797-803(2005)). All mice were on a mixed 129×C57BL6×FVB genetic background.Animals were housed in a pathogen-free barrier environment throughoutthe study. Experimental procedures involving the use of laboratory micewere reviewed and approved by the appropriate committee. GraphPad Prismsoftware was used for generating survival curves and for statisticalanalyses.

Cell Culture

Bone marrow cells were obtained by flushing of tibia and femur bones of2-month-old WT:INK-ATTAC transgenic mouse lines and cultured asdescribed elsewhere (Soleimani and Nadri, Nat. Protoc., 4:102-106(2009)). In brief, after washing by centrifugation at 400×g for 10minutes and counting of viable cells with trypan blue, cells wereresuspended in DMEM containing 15% FBS to a final concentration of 5×10⁶viable cells per mL. Initially, cells were plated in 6-well tissueculture dishes at 3.5 mL/well (1.9×10⁶ cells/cm²). Cultures were kept ina humidified 5% CO₂ incubator at 37° C. for 72 hours, when non-adherentcells were removed by changing the medium. Assays were performed oncells that had been trypsinized and seeded to confluency in 24-wellplates. To induce senescence and evaluate expression of the INK-ATTACtransgene, cells were treated with 1 μM rosiglitazone (Cayman ChemicalCompany, Ann Arbor, Mich.) or with vehicle. The accumulation ofGFP-positive cells was observed by fluorescence microscopy. After 5 daysof rosiglitazone treatment, cells were then washed with PBS and treatedwith vehicle, 1 μM rosiglitazone, 10 nM AP20187, or both. After 48hours, cultures were fixed and stained for SA-fl-Gal activity asdescribed elsewhere (Dimri et al., Proc. Natl. Acad. Sci. USA,92:9363-9367 (1995)).

qRT-PCR and Flow Cytometry

RNA extraction, cDNA synthesis, and qRT-PCR from whole-mouse tissue wereperformed as described elsewhere (Baker et al., Nat. Cell Biol.,10:825-836 (2008)). To perform qRT-PCR on GFP⁺ and GFP⁻ cell populationsof IAT, single-cell suspensions of stromal vascular fraction wereprepared from about 50 mg IAT as described elsewhere (Kirkland et al.,Int. J. Obes. Relat. Metab. Disord., 20(Suppl 3):5102-107 (1996)). GFP⁺and GFP⁻ cells were then separated and collected using a FACS Aria CellSorter running FACSDiva software (BD Biosciences). RNA was extractedfrom these cells using an RNeasy Micro Kit (Qiagen), and cDNAsynthesized using a WT-Ovation RNA Amplification kit (NuGENTechnologies, Inc.) according to the manufacturers' protocols.

qRT-PCR primers were as follows: FKBP-Casp8 forward,GAATCACAGACT-TTGGACAAAGTT (SEQ ID NO:25); FKBPCasp8 reverse,GGTCAAAGCCCCT-GCATCCAAG (SEQ ID NO:26); EGFP forward,CAAACTACAACAGCCACAACG (SEQ ID NO:27); and EGFP reverse,GGTCACGAACTCCAGCAG (SEQ ID NO:28). Sequences of other primers used wereas described elsewhere (Baker et al., Nat. Cell Biol., 10:825-836(2008)). Statistical differences were determined using two-tailedunpaired t tests.

Analysis of Progeroid Phenotypes

Bi-weekly checks for lordokyphosis and cataracts were performed asdescribed elsewhere (Baker et al., Nat. Cell Biol., 10:825-836 (2008)).Skeletal muscle fiber diameter measurements were performed on crosssections of gastrocnemius and abdominal muscles of female mice (n=6 miceper genotype). Fifty total fibers per sample were measured using acalibrated computer program (Olympus MicroSuite Five). Fat cell diametermeasurements were performed on IAT according to the same method.Dissection, histology, and measurements of dermal and adipose layers ofdorsal skin were performed as described elsewhere (Baker et al., Nat.Genet., 36:744-749 (2004)). Measurements of body weight, length,gastrocnemius muscle, and assorted adipose deposits were performed on8-10-month-old females (n=6 per genotype). Bone mineral content, bonemineral density, and total body adipose tissue were analyzed by DEXAscanning as described elsewhere (Krishnamurthy et al., J. Clin. Invest.,114:1299-1307 (2004)) (n=6 per genotype). Exercise measurements wereperformed on 8-10-month-old mice as described elsewhere (Handschin etal., J. Biol. Chem., 282:30014-30021 (2007); and LeBrasseur et al., J.Gerontol. A. Biol. Sci. Med. Sci., 64:940-948 (2009)). Animals wereacclimated for three days for 5 minutes at a speed of 5 m/minute priorto experimentation. For the experiment, the speed of the treadmill beganat 5 m/minute and was increased to 8 m/minute after 2 minutes.Thereafter, the speed was increased at a rate of 2 m/minute every 2minutes, and the time (in seconds) and distance (in meters) toexhaustion, as defined by an inability to move along the treadmill withstimulation, were determined. The formula to determine the amount ofwork (J) performed was: mass (kg)*g (9.8 m/s²)*distance (m)*sin(θ) (withan incline of θ=5°).

In Vivo BrdU Incorporation and SA-β-Gal Staining

Analyses for in vivo BrdU incorporation were performed in 8-10-month-oldfemale mice (n=6 per genotype) as described 13. Adipose tissue depotswere stained for SA-β-Gal activity as described elsewhere (Baker et al.,Nat. Cell Biol., 10:825-836 (2008)).

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A transgenic mouse, the nucleated cells of whichcontain a transgene, said transgene containing a promoter sequenceoperably linked to a nucleic acid sequence encoding a polypeptide havingthe ability to kill a cell or facilitate the killing of a cell when saidtransgenic mouse is administered a compound, wherein senescent cells ofsaid transgenic mouse express said polypeptide, and wherein saidsenescent cells of said transgenic mouse are killed when said compoundis administered to said transgenic mouse.
 2. The transgenic mouse ofclaim 1, wherein less than 10 percent of non-senescent cells of saidtransgenic mouse are killed when said compound is administered to saidtransgenic mouse.
 3. The transgenic mouse of claim 1, wherein saidpromoter sequence is a p16^(Ink4a) promoter sequence.
 4. The transgenicmouse of claim 1, wherein said polypeptide comprises a caspase 8polypeptide sequence.
 5. The transgenic mouse of claim 1, wherein saidpolypeptide comprises a FKBP polypeptide sequence.
 6. The transgenicmouse of claim 1, wherein said polypeptide is a FKBP-caspase 8 fusionpolypeptide.
 7. The transgenic mouse of claim 1, wherein said compoundis AP20187.
 8. The transgenic mouse of claim 1, wherein the geneticbackground of said transgenic mouse is a BubR1^(H/H) genetic background.9. The transgenic mouse of claim 1, wherein said transgene comprisesnucleic acid encoding a marker polypeptide.
 10. The transgenic mouse ofclaim 9, wherein said marker polypeptide is a GFP polypeptide.